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Abstract

Glutamatergic synapses play critical roles in brain functions and diseases. Long-term
potentiation (LTP) is a most effective cellular model for investigating the synaptic
changes that underlie learning as well as brain disease – although different molecular
mechanisms are likely involved in LTP in physiological and pathological conditions.
In the case of learning, N-methyl-D-aspartate (NMDA) receptor is known to be important
for triggering learning-related plasticity; alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
(AMPA) receptors are thought to be important for the expression of synaptic changes.
In this review, I will examine recent evidence on the novel roles of NMDA receptors,
in particular NR2B subunit-containing NMDA receptors in learning and chronic pain.
A positive feedback control of NR2B receptor subunit is proposed to explain cortical sensitization involved
in chronic pain, but not learning and memory.

Introduction

The NMDA receptor acts as an activity-dependent coincidence detector in the central
nervous system (CNS). The majority of past research has focused on the synaptic changes
– namely presynaptic changes in glutamate release, and postsynaptic AMPA receptor
changes – following the activation of the NMDA receptor. Few review articles are available
about the potential long-term plastic changes in NMDA receptor subunits, in particular
NR2B subunit. In this review, by focusing on physiological (memory) and pathological
(chronic pain) functions, I will examine previous and recent evidence for long-term
plastic changes of NMDA receptor NR2B subunit in the central sensory and 'learning'
synapses, as well as the molecular machinery that may contribute to NMDA receptor
NR2B subunit trafficking and postsynaptic insertion.

LTP as a cellular model for brain plasticity

It is well known that central synapses are highly plastic, and long-term changes in
synaptic transmission contribute to different functions of the brain throughout the
lifespan. Two major forms of synaptic plasticity have been widely investigated: long-term
potentiation (or called LTP) and long-term depression (or called LTD). While LTP can
enhance synaptic functions in certain regions of the brain, LTD attenuates or reduces
the efficacy of synaptic transmission. Such biphasic synaptic plasticity is not limited
at excitatory, glutamatergic synapses. Both LTP and LTD have been also reported in
inhibitory synapses, and underlying cellular and molecular mechanisms are different.
Recent studies using different induction protocols reveal that the mechanisms for
central LTP are likely to be different, depending the induction protocols, regions
of the CNS, input fibers and postsynaptic neurons recorded [1-5]. There is no doubt that many different molecular targets will be continuously revealed
in future, one urgent task is to verify the physiological or pathological relevant
of synaptic LTP/LTD induced by experimental induction protocols. Furthermore, new
forms of LTP and LTD remain to be discovered to mimic physiological and/or pathological
changes under in vivo conditions (e.g., presynaptic enhancement of neurotransmitter release after tissue
injury in the anterior cingulate cortex (ACC) (see [6]).

What has been recognized about the potential functions of LTP is its contribution
to many key brains functions in addition to learning and memory [7-11]. At the spinal cord dorsal horn where the first sensory synapses are located, LTP
of sensory synaptic transmission can be induced by different experimental protocols
[12-14] or peripheral injury [14]. Potentiated excitatory synaptic transmission is believed to contribute to spinal
sensitization that at least in part attributes to behavioral hyperalgesia and allodynia
during chronic pain. In the basolateral amygdala, LTP can be induced between thalamic/cortical
inputs and postsynaptic principle neurons [15] or fear conditions [11], such enhanced responses are important for encoding fearful information. In the hippocampal
CA1 region where most of LTP studies have been reported, LTP can be induced and reliable
detected, even with field recording electrodes [7,8]. However, despite a huge amount of literature on hippocampal LTP, it remains to be
demonstrated that if a simple spatial training trial may induce LTP in certain population
of CA1 neurons.

Finally, in the prefrontal cortical (PFC) neurons including the ACC, LTP is induced
by the pairing, spike-timing and theta burst protocols [16] as well as peripheral injury [17,6]. It has been proposed that the injury caused synaptic potentiation contribute to
chronic pain and pain-related high brain functions including fear and emotion [18]. Therefore, it is clear that studying central LTP provides fundamental mechanisms
for brain functions – from pain transmission to fear and chronic pain.

NMDA receptor and LTP

One major feature of LTP is the requirement of activation of NMDA receptors. As compared
with downstream signaling protein inhibitors, the inhibition of NMDA receptor by bath
application of AP-5 reliably blocks the induction of LTP. In most cases, basal synaptic
responses are not affected the same application of AP-5, indicating the selective
roles of NMDA receptors in the induction. The key mechanism for the involvement of
NMDA receptor in the induction of LTP is its voltage-dependence. At resting membrane
potentials, NMDA receptors are inactive due to pore blockade by extracellular Mg2+, even in the presence of glutamate. Thus, in order to activate NMDA receptors at
synapses, two events need to happen simultaneously. First, glutamate needs to be released
and binds to NMDA receptors; second, the postsynaptic membrane needs to be depolarized
so that the block by extracellular Mg2+ can be removed. NMDA receptor-mediated calcium influx from the extracellular space
into the postsynaptic cells then activates a series of signaling molecules within
postsynaptic cells, including protein kinases, protein phosphatases, immediate early
genes (i.e., genes can be activated rapidly, or called third messengers), as well
as enzymes producing diffusible retrograde messengers. Although the requirement for
NMDA receptor in LTP is easily found, application of NMDA did not cause synaptic potentiation
[7], making biochemical studies of LTP difficult.

The requirement of the activation of NMDA receptors in synaptic LTP is common among
many brain regions. In the spinal cord dorsal horn neurons, LTP of AMPA receptor mediated
EPSCs were induced by the induction protocol that paired synaptic stimulation (2 Hz)
with postsynaptic depolarization (+30 mV) [12]. In the amygdala, LTP induced by the pairing protocol is blockaded by NMDA receptor
antagonist AP-5 [19]. In the ACC, bath application of a NMDA receptor antagonist AP-5 completely abolished
the induction of LTP induced by different induction protocols in the pyramidal cells
[16], indicting that the induction of ACC LTP is completely dependent of postsynaptic
activation of NMDA receptors.

NMDA receptor independent LTP

NMDA receptor independent LTP have been also reported. Most of these experiments showing
NMDA receptor independent LTP are performed in the presence of a NMDA receptor blocker.
It has to be noted that some experiments only used a sub-dose of AP-5. It is critical
to demonstrate by whole-cell patch-clamp recording that the same dosage of NMDA receptor
antagonist does in fact completely eliminate NMDA receptor mediated excitatory postsynaptic
currents (EPSCs) [16]. Depending on the regions of the CNS and the stimulation protocols, there are many
reports of NMDA receptor as an independent form of LTP. Several neurotransmitter receptors
or ion channels have been implicated in the initiation of LTP, such as L-type voltage
gated calcium channels (L-VGCCs), metabotropic glutamate receptors (mGluRs), serotonin
receptors, dopamine receptors and kainate (KA) receptors. For example, in the hippocampus,
in the presence of AP-5, very strong tetanic stimulation or bath application of TEA
induced LTP that is sensitive to the blockade of L-VGCCs [20,21]. Bath application of tACPD, an agonist of mGluRs, produced long-lasting potentiation
that may require cGMP-related signaling pathways [22]. In a recent study using gene knockout of KA receptor subtype 6 (KA GluR6) mice,
the pairing induced LTP was reduced or blocked in the ACC and amygdala, suggesting
that KA receptor may contribute to synaptic potentiation [23].

Composition of NMDA receptors

Functional NMDA receptors contain heteromeric combinations of the NR1 subunit plus
one or more of NR2A-D. While NR1 distributes ubiquitously in the CNS, NR2 subunits
exhibit regional distribution, and the amount of expression is developmentally related.
In the neonatal brains, NR2B and NR2D subunits are highly expressed, and over the
course of development, they are substituted or replaced by NR2A and NR2C. In humans
and rodents, NR2A and NR2B subunits predominate in forebrain structures [24,25]. NR2A and NR2B subunits confer distinct properties to NMDA receptors; heteromers
containing NR1 plus NR2B mediate a current that decays three to four times more slowly
than receptors composed of NR1 plus NR2A [25-27]. Unlike other ionotropic channels, NMDA receptors are 5–10 times more permeable to
calcium, a critical intracellular signaling molecule for triggering postsynaptic and
possible presynaptic plastic changes, than to Na+ or K+. NMDA receptor mediated currents are long-lasting compared with the rapidly desensitizing
kinetics of AMPA and KA receptor channels.

Contribution of NR2B-containing NMDA receptors to synaptic LTP

The requirement of NR2B-NMDA receptors in synaptic potentiation has been reported
in different areas of the CNS (see Table 1). Considering the NMDA receptor mediated currents are largely carried out by NR2A-containing
NMDA receptors, one expects that inhibition of NR2B receptor alone may not be sufficient
to produce complete blockade of synaptic potentiation. Indeed, in the ACC synapses,
bath application of selective NMDA receptorNR2B antagonists significantly reduced
but not blocked the induction of LTP by the pairing protocol [16]. Similarly, in the lateral amygdala, NMDA receptor NR2B antagonist Ro 25–6981 significantly
reduced NMDA receptor-dependent LTP induced by a pairing protocol [28]. In the hippocampal CA1 region, some studies have reported that the activity of NR2B-NMDARs
is not required for synaptic potentiation or LTP [29]. However, a previous study using genetic overexpression of NR2B subunits indicate
the involvement of NMDA NR2B receptor. In the hippocampus of transgenic mice with
NMDA NR2B overexpression, LTP induced by tetanic stimulation or repetitive stimulation
were significantly enhanced as compared with wild-type littermates [30] (Figure 1).

Considering that NR2B- and NR2A- NMDA receptors have different biophysical properties
and couple to different intracellular signaling cascades, it may be possible that
different induction protocols may activate different NMDA receptor subtypes. It has
been reported that different LTP-inducing protocols recruit different signaling pathways.
For example, in the amygdala, pairing-protocol induced LTP depends on L-VGCCs but
not NMDA receptors, while tetanus-stimulation induced LTP involves NMDA receptor but
not L-VGCCs [31]. Recently, we have shown that the involvement of NR2B-NMDA receptors in hippocampal
LTP are dependent on induction protocols [32,33]. NR2B-NMDA receptors are required for LTP induced by the spike-timing protocol, but
not by the pairing protocol [5]. The same dose of Ro 25–6981 reduced LTP induced by the spike-timing protocol, but
not by the pairing protocol. Interestingly, neither LTP induced by two train tetanic
stimulation nor late-phase LTP induced by multiple train stimulation was affected
by NR2B antagonist Ro 25–6981. Furthermore, calcium imaging studies showed that the
NR2B-NMDA receptor mediated Ca2+ transients were faster under the spike-timing than pairing protocols, which might
explain the different significance of NR2B-NMDARs in LTP under the two protocols since
fast Ca2+ transients are better for LTP, but slow Ca2+ transients not [5].

Requirement of NMDA receptor NR2B subunit in behavioral learning

Although the NMDA NR2B receptor antagonists have been available for a few years, there
are a few studies that have investigated the contribution of NMDA NR2B receptors to
behavioral learning. For spatial water maze memory, it has been reported that 60-min
pretreatment with (+/-)-CP-101,606 (60 mg/kg, p.o.), a dose that fully occupied hippocampal
NR1/NR2B subunit-containing receptors, as determined by ex vivo NMDA receptor-specific
[3H]ifenprodil binding immediately following water maze experiments, had no effect on
acquisition or the probe trial. These results suggest that antagonists selective for
NR1/NR2B subunit-containing receptors may not impair spatial memory in rats in the
Morris water maze [34].

Unlike spatial memory, the contribution of NMDA NR2B receptors to fear memory has
been reported. Systemic injections of NMDA NR2B receptor antagonist ifenprodil before
training led to a dose-dependent impairment in the acquisition of auditory and contextual
fear conditioning, whereas injections before testing had no effect [35]. Recently, similar administration of NMDA receptor NE2B subunit antagonist Ro 25–6981
significantly attenuated fear extinction but not re-extinction recall [36]. In the hippocampal CA1 region, pre-training intra-CA1 infusion of ifenprodil or
Ro 25–6981 impaired the contextual fear memory induced by five CS-US pairings, with
no effect on the memory induced by one conditioned stimulus (CS)-unconditioned stimulus
(US) pairing [5]. These findings are in good accord with previous genetic studies that forebrain NR2B
overexpression enhanced spatial and fear memory [30]. Furthermore, intra-amygdala infusions of ifenprodil mirrored systemic injection
results and, in addition, showed that the effects are attributable to a disruption
of fear learning rather than a disruption of memory consolidation. NMDA receptors
in lateral amygdala are thus involved in fear conditioning, and the NR2B subunit appears
to make unique contributions to the underlying plasticity [35]. In addition to the amygdala, a recent study indicates that neurons in the ACC may
also contribute to the formation of fear memory. Inhibition of NMDA NR2B functions
in the ACC by local microinjection of pharmacological NR2B antagonists or NR2B siRNA
manipulation significantly reduced fear memory [16], suggesting that cortical NMDA receptor NR2B subunit also contribute to fear memory
formation.

NMDA NR2B receptor does not undergo potentiation in memory storage

Unlike AMPA receptors during early phase memory, it is generally believed that NMDA
receptors do not undergo rapid and prolonged changes during memory (or hippocampal
LTP) (Table 2). Systemic administration of NMDA NR2B antagonists after the training and before
the testing did not affect fear memory [35]. Consistent with this finding, AMPA receptor mediated responses are found to be enhanced
after fear conditioning in the amygdala and NMDA-mediated transmission in the thalamic-to-lateral
amygdala pathway is not facilitated after fear conditioning. Western blots show a
reduction in phosphorylated-NR1, NR2A, and NR2B subunit protein expression in the
amygdala from fear-conditioned animals. There are at least three possible physiological
significances for the reduction in NMDA receptor NR2B functions [37]. First, the down-regulation of the NMDA receptor may protect against neuronal excitotoxicity
of unchecked NMDA receptor recruitment during the induction and consolidation of fear
memories. It is well known that NMDA NR2B receptor antagonists have neuroprotective
effects. Second, reduced NMDA current and protein may allow persistence of the "capacity
to reactivate" amygdala pathways for the induction of future fear memories. The overexcitation
caused by the upregulation of NMDA receptors may prevent the formation of new fear
memory that is critical for animals to survive in the natural environment. Finally,
a persistent long-term depression of NMDA transmission may occur after fear learning
[37]. Similar results have been found in the visual cortex. Experience-dependent plasticity
is reported to cause changes in the NR2A:NR2B ratio, favoring the expression of NMDA
receptor NR2A subunit [38-41].

Upregulation of NMDA receptor NR2B subunit functions in chronic pain

In NMDA receptor NR2B subunit genetically overexpression mice, we found that chronic
pain – but not acute or physiological pain – was selectively enhanced [30,42] (see Figure 2), providing the first genetic evidence that forebrain NMDA NR2B receptor is critical
for chronic pain. This finding also offers additional reasons to explain why NMDA
NR2B receptor is not undergoing upregulation during learning (see above). Does genetically
overexpression of NR2B mimic physiological or pathological conditions? In a recent
study we found that after persistent inflammation (the Complete Freund's Adjuvant
(CFA) animal model for chronic inflammation), the expression of NMDA NR2B receptors
in the ACC was increased over a long-period of time, thereby increasing the NR2B component
in NMDA receptor mediated EPSCs [17] (see Figure 3A, B). In the behavioral allodynia test, microinjection into the ACC or systemic administration
of NMDA NR2B receptor with selective antagonists inhibited behavioral responses to
peripheral inflammation [17]. These results are consistent with genetic studies that mice with NMDA receptor NR2B
subunit forebrain overexpression selectively enhanced inflammation-related persistent
pain without significant changes in acute pain [43]. The anti-allodynic effects of NMDA NR2B receptor antagonists have been also reported
in other animal models of chronic pain. We believe that these findings provide direct
evidence that NMDA NR2B receptors undergo long-term plastic changes in the brain after
injury.

Figure 2.Selective enhancement of chronic pain in 'smart' mice with NR2B overexpression in
forebrains. a. Overexpression of NR2B mRNA in pain-related forebrain areas including the ACC and
insular cortex (IC). No overexpression of NR2B was detected in the spinal cord. b. Enhancement of behavioral nociceptive licking responses to peripheral subcutaneous
injection of formalin in NR2B transgenic mice. c. Summarized three different phases of behavioral nociceptive licking responses in
wild-type and NR2B transgenic mice. Similar increases in behavioral nociceptive responses
were found in the second line of NR2B transgenic mice. Modified from Wei et al. (2001).

Figure 3.Peripheral inflammation triggers long-term increases in NMDA NR2B receptor mediated
currents in cingulate pyramidal cells. NR2B sensitive component of NMDA receptor mediated EPSCs were enhanced in mice with
CFA injection. a. Traces of EPSCs show the currents at different time points during application of
drugs. Ro 25–6981 produced its maximal effect at 3 min after bath application, and
a higher dose of Ro 25–6981 (3 μM) had no additional effects. The remaining currents
can be totally blocked by AP-5 (50 μM). b. A selective NR2B antagonist, Ro 25–6981, partially inhibited NMDA receptor-mediated
EPSCs. The time course of changes in EPSC amplitude before and during the application
of Ro 25–6981 (0.3 and 3 μM) and AP-5 (50 μM) in ACC neurons from both saline (control,
open symbols) and CFA-injected (filled sumbols) mice is shown. Modified from Wu et
al. (2005).

Recent studies in other central synapses suggest that NMDA NR2B receptor up-regulation
is likely to be reliant on activity-dependent mechanisms. The molecular motor protein
KIF 17 has been shown to be involved in the active transport of NMDA receptor NR2B
subunits [44-46]. NR2B contains a cAMP response element-binding protein (CREB) binding domain which
may couple increases in intracellular calcium with the increase in NR2B expression.
Since NMDA receptors play an important role in activity-dependent plasticity in the
ACC, we suggest that NMDA NR2B subunit may be regulated through NMDA-calcium-CaM-dependent
signaling pathways. The activation of NMDA receptors triggers postsynaptic calcium,
leading to the activation of calcium-stimulated CREB in the ACC after peripheral or
central injury [42] (see Figure 4).

Figure 4.Central signaling pathways contribute to chronic pain. In the ACC, glutamate is the major fast excitatory transmitter between input fibers
and pyramidal cells. Peripheral injury such as tissue inflammation or nerve injury
trigger a burst of abnormal activity in the ACC circuits; and subsequently activate
postsynaptic NMDA receptors on cingulate pyramidal cells located in layer II-III.
Activation of NMDA receptor triggers calcium influx. In adult ACC pyramidal cells,
most of NMDA receptors are the combination of NR1-NR2A, NR1-NR2B with possible minor
component of currents made of NR1-NR2A-NR2B. Postsynaptic increases in Ca2+ leads to activation of Ca2+-calmodulin (CaM) dependent pathways. Among them, Ca2+ and CaM stimulated AC1 is activated, and this activation leads to the generation of
the key second messenger cAMP. Subsequently, cAMP activates PKA. PKA then translocates
to the nucleus and phosphorylates CREB. NR2B contains a CREB binding domain which
may couple increases in intracellular calcium with the increase in NR2B expression.
Subsequently, postsynaptic synthesis of NMDA NR2B is increased, and together with
endogenous motor protein KIF17, these new NR2B subunits are added to postsynaptic
NMDA receptors. Such positive feedback control may further enhance neuronal excitability within the ACC, and contribute to chronic
pain.

cAMP as a key second messenger

Studies using gene knockout of calcium-calmodulin dependent adenylyl cycles AC1 and
AC8 revealed that AC1 pays key roles in triggering injury related central plasticity.
In AC1 knockout mice, LTP, a likely synaptic model for chronic pain in the cortex,
was completely abolished [47]. The effects of inhibition of AC1 are selective as both basal synaptic responses
(mostly mediated by AMPA receptor) and NMDA receptor mediated responses were not affected
[47,18]. Because of the ACC LTP induced by the paring protocol is mediated by postsynaptic
AMPA receptor (likely through postsynaptic receptor trafficking) [48], it is suggested that AC1 activity is critical for calcium-triggered AMPA receptor
trafficking. Consistent with in vitro slice findings, behavioral responses in different
animal models of chronic pain are significantly reduced or blocked in AC1 knockout
mice.

Recent studies using the animal model of neuropathic pain actually show that postsynaptic
AMPA receptor mediated responses are enhanced in the ACC synapses of mice receiving
nerve injury. The enhancement of AMPA receptor mediated EPSCs is likely through the
phosphorylation of postsynaptic AMPA receptor GluR1 subunit at PKA site [6]. Such nerve injury triggered enhancement of postsynaptic responses and GluR1 receptor
phosphorylation are abolished in AC1 knockout mice, suggesting that AC1 dependent-cAMP
pathway is important. In addition to postsynaptic mediated effects, presynaptic enhancement
of glutamate releases in the ACC synapses have been also found, and AC1 is also required
for triggered these presynaptic changes [6]. It remains to be investigated if such AC1 contribution is directly on presynaptic
terminals, or act through possible retrograde messengers in the ACC [18].

Studies using AC1 knockout mice also demonstrated that AC1 activity is critical for
NR2B upregulation after the injury. Changes in NMDA NR2B antagonist sensitive EPSCs
caused by the nerve injury were abolished in mice lacking AC1 (unpublished data).
It is possible that NMDA NR2B receptor-AC1-cAMP-CREB-NR2B form a positive cellular
feedback to reinforce the NMDA receptor functions in the ACC neurons. Together with
NMDA receptor mediated AMPA receptor potentiation, these synaptic changes greatly
enhance excitatory synaptic transmission in the ACC after the injury, and thus contribute
to cortical pain (see Figure 4).

Based on the discovery of NMDA NR2B receptor functions in LTP and behavioral memory,
there are several proposals of developing NMDA NR2B function enhancer to increase
memory functions in so-called low IQ adults or to rescue the memory loss in the patients.
This hypothesis gains some supports from the experiments of the environment enrichment
in that both NMDA NR2B functions and behavioral learning are significantly enhanced.
However, considering the negative finding of NMDA NR2B receptors in the expression
of memory, it is unlikely that enhancing NMDA NR2B functions will be beneficial for
maintaining newly formed memory (see above). Furthermore, enhancing NMDA NR2B functions
may enhance chronic pain in case of any tissue or nerve injury [18]. In addition to chronic pain, the potential roles of NMDA NR2B receptors in cell
death, seizure have been reported. Enhancing NR2B receptors may also enhance these
potential risks.

To use the NMDA receptor NR2B antagonists to control brain disease and chronic pain
have been reported by many groups. For example, in the case of chronic pain, it has
been reported that NR2B antagonists produced powerful analgesic effects in animal
models of chronic pain (see above). The action off site for NR2B antagonist is mainly
due to cortical NR2B receptors, although it may also exert some of its effects in
the spinal cord level. Alternatively, in the case that side effects in humans are
found with NR2B antagonists, one may target on downstream proteins such as AC1. In
summary, we are just beginning to understand how central synapses may undergo plastic
changes during learning or after peripheral tissue injury, understanding basic mechanisms
for such long-term plasticity may help us to design better medicines for treating
memory loss and chronic pain.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

Supported by grants from the Canadian Institutes of Health Research (CIHR81086 and
84256), the EJLB-CIHR Michael Smith Chair in Neurosciences and Mental Health, and
the Canada Research Chair to Dr. Min Zhuo.